Protein fragment complementation assay (PCA) for the detection of protein-protein, protein-small molecule and protein nucleic acid interactions based on the E. coli TEM-1 β-Lactamase

ABSTRACT

The present invention describes an assay method comprising: (A) generating (1) at least a first fragment of a reporter molecule linked to a first interacting domain and at least a second fragment of a reporter molecule linked to a second interacting domain, or (2) nucleic acid molecules that code for (A)(1) and subsequently allowing said nucleic acid molecules to produce their coded products; then, (B) allowing interaction of said domains; and (C) detecting reconstituted reporter molecule activity, where said reporter molecule can react with a penicillin- or a cephalosporin-class substrate.

This Application claims the benefit of U. S. Provisional Application No.60/208,485 filed Jun. 2, 2000, the entire contents of which are herebyincorporated by reference. This Application is also acontinuation-in-part U.S. Ser. No. 09/499,464 filed Feb. 7, 2000; andnow U.S. Pat. No. 6,428,951; which is a continuation of U.S. Ser. No.09/017,412 filed Feb. 2, 1998; and now U.S. Pat. No. 6,270,964. Theentire contents of all those patents and applications are incorporatedby reference herein.

TECHNICAL FIELD OF THE INVENTION

The present invention relates generally to protein complementationassays (PCA) and more specifically to PCA assays based on the E. coliTEM-1 β-Lactamase for the detection of protein-protein, protein-smallmolecule and protein nucleic acid interactions.

BACKGROUND OF THE INVENTION

Applicants' have previously described oligomerization domain-assistedcomplementation of enzyme fragments as a general strategy for detectingprotein-protein, protein small molecule and protein nucleic acidinteractions (ref. 9). In the present invention, we describe assaysbased on the E. coli TEM-1 β-Lactamase (Accession number: AAB59737). Inthe present invention, applicants' disclose three assays in mammaliancells: (1), an in vitro colorimetric assay using the substratenitrocefin, and (2) an in vivo positive/negative fluorescence assayusing the substrate CCF2/AM. The invention is also directed to positiveand negative survival assays using cephalosporin-cytotoxic pro-drugconjugates, as well as a series of β-lactamase point mutations thatwould be predicted to enhance the efficiency of the β-lactamase PCA.

The TEM-1 β-lactamase is a member of a family of bacterial enzymes thathydrolyze antibiotics of the penicillin and cephalosporin class, thusimparting resistance to bacteria expressing these enzymes. TEM-1β-lactamase is the standard ampicillin resistance gene included in mostplasmids used in molecular biology. The three-dimensional structure,proposed catalytic mechanism and optimal substrates and inhibitors havebeen well documented. TEM-1 β-Lactamase is a small (29 kiloDaltons) andmonomeric protein consisting of 286 amino acids. The first 23 aminoacids constitute a secretory signal peptide. β-lactamases catalyses theirreversible hydrolysis of the amide bond of β-lactam rings inpenicillin or cephalosporin compounds. β-lactamases are secreted intothe periplasmic space of gram-negative strains or into the outer mediaby their gram-positive counterparts where they normally act. However,they will accumulate in the cytoplasm when expressed in E. coli or otherprokaryotic or eukaryotic cells if the secreting signal peptide isgenetically deleted, without effecting catalytic activity.

TEM-1 β-lactamase meets all of the essential criteria to be an excellentcandidate for a PCA strategy. Specifically, TEM-1 β-lactamase is arelatively small, monomeric protein and is well characterized bothstructurally and functionally. TEM-1 β-lactamase can be expressed in andis not toxic to prokaryotes and eukaryotes. In addition to these, uniquefeatures include that: First, β-lactamase is strictly a bacterial enzymeand has been genetically deleted from many standard E. coli strains. Itis not present at all in eukaryotes. Thus, a β-lactamase PCA could beused universally in eukaryotic cells and many prokaryotes, without anyintrinsic background. Second, assays are based on catalytic turnover ofsubstrates with rapid accumulation of product. This enzymaticamplification should allow for relatively weak molecular interactions tobe observed. Finally, the assay can be performed simultaneously orserially in a number of modes, such as in vitro colorimetric orfluorometric assays, or in vivo fluorescence or survival assays. Assayscan be performed independent of the measurement platform and can easilybe adapted to high-throughput formats requiring only one pipetting step.

The PCA strategy of the present invention is based on the reassembly oftwo rationally designed complementary fragments of TEM-1 β-lactamase.Crystal structures of TEM-1 suggest that residues 196-200 form a loopsituated outside of the core of the protein and distal to the enzymaticpocket (FIG. 1). This loop is not implicated in the catalytic machineryand seems not to be important for catalysis (ref. 4). For these reasons,this site was selected to generate the two fragments. We chose to cut inthe middle of the loop between residues Glu197 and Leu198. In addition,the secreting signal peptide of 23 amino acids was deleted to leave onlythe functional enzyme. Thus fragment [1] (BLF[1]) consists of residues24 to 197 and fragment [2] (BLF[2]) of residues 198-286. Each of thesefragments are linked to interacting domains (GCN 4 leucine Zipper or thepair of rapamycin inducible interacting proteins FKBP/FRB domain) by alinker of 15 amino acids (Gly-Gly-Gly-Gly-Ser)₃.

OBJECTS OF THE INVENTION

A primary object of the present invention are protein complementationassays (PCA) based on the E. coli TEM-1 β-lactamase.

Another object of the present invention is an in vitro colorimetric PCAassay in mammalian cells using the substrate nitrocefin.

A further object of the invention is an in vivo positive/negativefluorescence PCA assay in mammalian cells using the substrate CCF2/AM.

Still, another object of the invention is positive and negative survivalassays using cephalosporin-cytotoxic pro-drug conjugates.

An additional object of the invention is positive and negative survivalassays using a series of β-lactamase point mutations that would bepredicted to enhance the efficiency of the β-lactamase PCA.

We have found that these objects and others are achieved by the use ofE. coli TEM-1 β-Lactamase as further described below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates technical information about the enzymes of theinvention as well as the 3-D structure (ref. 7) of β-lactamase.

FIG. 2A shows the chemical structure of Nitrocefin and mechanism ofhydrolysis of β-lactams.

FIG. 2B describes the visualization of positive and negative controlswherein Negative: yellow substrate, red: hydrolyzed product ofnitrocefin.

FIG. 3 illustrates GCN4 Zipper interactions in HEK 293 cells wherein (A)are untransfected cells, (B) are cells transfected with wild-type TEM-1β-lactamase, (C) is the Negative Control Cells transfected with noninteracting pairs of protein FRB-5a.a.-BLF[1] and GCN4Zip-5a.a.-BLF[2],and (D) are the Positive Control Cells transfected with interactingpairs of protein GCN4Zip-5a.a.-BLF[1]and GCN4Zip-5a.a.-BLF[2].

FIG. 4 describes FKBP and FRB interactions in HEK 293 cells wherein (A)is the structure of the interacting pair of FKBP and FRB domain inducedby rapamycin, (B) is the quantitation of the dose-response inducedinteraction of FRB-5a.a.-BLF[1] and FKBP-5a.a.-BLF [2] with rapamycinconcentration ranging from 0.15 nM to 0.3 μM. Inhibition of the FKBP/FRBdomain interaction with FK506 concentration ranging from 6.9 nM to 15 μMagainst 50 nM rapamycin, (C) is the dose response induced interactioncurve with rapamycin concentration ranging from 0.15 nM to 0.3 μM(Determined K_(d) of 3 nM), and (D) is the inhibition curve of 50 nMrapamycin induced interaction between FKBP/FRB domain with FK506concentration ranging from 6.9 nM to 15 μM.

SUMMARY OF THE INVENTION

The instant invention is directed to an assay method comprising: (A)generating: (1) at least a first fragment of a reporter molecule linkedto a first interacting domain and at least a second fragment of areporter molecule linked to a second interacting domain, or (2) nucleicacid molecules that code for (A)(1) and subsequently allowing saidnucleic acid molecules to produce their coded products; then, (B)allowing interaction of said domains; and (C) detecting reconstitutedreporter molecule activity, where said reporter molecule can react witha penicillin- or cephalosporin-class substrate.

The invention is also directed to an assay method comprising: (A)exposing a host cell to: (1) at least a first fragment of a reportermolecule linked to a first interacting domain and at least a secondfragment of a reporter molecule linked to a second interacting domain;or (2) compounds that code therefor; and (B) detecting reconstitutedreporter molecule activity, where a reporter molecule and a host cellare used that yield a signal essentially without any intrinsicbackground.

In the present invention, there is also described an assay methodcomprising: (A) exposing a host cell to: 1) at least a first fragment ofa reporter molecule linked to a first interacting domain and at least asecond fragment of a reporter molecule linked to a second interactingdomain; or (2) compounds that code therefor; and (B) detectingreconstituted reporter molecule activity, where a reporter moleculesubstrate is added that becomes trapped within said cell after entrancetherein.

The invention is further directed to an assay method comprising: (A)exposing a host cell to: (1) at least a first fragment of a reportermolecule linked to a first interacting domain and at least a secondfragment of a reporter molecule linked to a second interacting domain;or (2) compounds that code therefor; and (B) detecting reconstitutedreporter molecule activity, where a reporter molecule substrate is addedthat has a fluorescent signal-producing system covalently associatedtherewith.

The present invention also describes an assay method comprising: (A)exposing a host cell to: (1) at least a first fragment of a reportermolecule linked to a first interacting domain and at least a secondfragment of a reporter molecule linked to a second interacting domain;or (2) compounds that code therefor; and (B) detecting host cellsurvival as an indication of reconstituted reporter molecule activity.

In another embodiment, the invention discloses an assay methodcomprising: (A) exposing a host cell to: (1) at least a first fragmentof a reporter molecule linked to a first interacting domain and at leasta second fragment of a reporter molecule linked to a second interactingdomain; or (2) compounds that code therefor; (B) further exposing saidcell to a compound to be assayed for its ability to interfere withinteraction of said first and second domains; and (C) detecting hostcell survival as an indication of interference with said interaction.

The invention also teaches a composition comprising a compound whichcomprises a fragment of an interacting domain linked to a fragment of areporter molecule that can hydrolyze either a penicillin class substrateor a cephalosporin class substrate.

In a further embodiment, the invention is directed to a compositioncomprising: (A) a first compound comprising a first fragment of aninteracting domain linked to a first fragment of a reporter moleculethat can hydrolyze either a penicillin class substrate or acephalosporin class substrate; and (B) a second compound comprising asecond fragment of an interacting domain linked to a second fragmentsaid reporter molecule.

The instant invention also describes an assay method comprising: (A)allowing at least two molecules capable of mutual interaction to drawinto close molecular proximity at least two reporter molecule fragmentswhich, when in close molecular proximity, form a complex capable ofreaction with a penicillin- or cephalosporin-class substrate; and (B)detecting a signal resulting from said reaction.

In a further embodiment, the invention describes an assay methodcomprising: (A) allowing at least two molecules capable of mutualinteraction to draw into close molecular proximity at least two reportermolecule fragments which, when in close molecular proximity, form acomplex capable of reaction with a penicillin- or cephalosporin-classsubstrate; and (B) detecting a signal resulting from said reaction,where there is essentially no intrinsic background in the assay.

The instant invention is also directed to an assay method comprising:(A) allowing at least two molecules capable of mutual interaction todraw into close molecular proximity at least two reporter moleculefragments which, when in close molecular proximity, form a complexcapable of reaction with a penicillin- or cephalosporin-class substrate;and (B) detecting a signal resulting from said reaction, where saidreaction occurs with a cell and said substrate becomes trapped withinsaid cell after entrance therein.

The invention further describes an assay method comprising: (A) allowingat least two molecules capable of mutual interaction to draw into closemolecular proximity at least two reporter molecule fragments which, whenin close molecular proximity, form a complex capable of reaction with apenicillin- or cephalosporin-class substrate; and (B) detecting a signalresulting from said reaction, where a reporter molecule substrate isadded that has a fluorescent signal-producing system covalentlyassociated therewith.

The invention further provides a cellular assay method comprising: (A)allowing at least two molecules capable of mutual interaction to drawinto close molecular proximity at least two reporter molecule fragmentswhich, when in close molecular proximity, form a complex capable ofreaction with a penicillin- or cephalosporin-class substrate; and (B)detecting cell survival as an indication of said reaction.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Substrates for Enzymatic Assay of β-lactamase

In practicing the instant invention two substrates have been used tostudy the β-lactamase PCA. The first one is the cephalosporinNitrocefin. This substrate is used in the in vitro colorimetric assay.β-lactamase is quite efficient for this substrate, having a kcat/km of17,000 mM⁻¹*S⁻¹ (ref. 4). Substrate conversion can be easily observed byeye; the substrate is yellow in solution while the product is a distinctruby red color. The rate of hydrolysis can be monitored quantitativelywith any spectrophotometer by measuring the appearance of red at 492 nm.Signal to background, depending on the mode of measurement can begreater than 30:1 (FIG. 2). As described below, this assay, at presentis performed with whole cell lysates, as nitrocefin is not membranepermeant. However, in principle addition of ester groups could besufficient to allow for membrane permeability and a true in vivocalorimetric assay could be performed.

We also performed an in vivo fluorometric assay using the substrateCCF2/AM. While not as good a substrate as nitrocefin (kcat/km of 1260mM⁻¹*S⁻¹) CCF2/AM has unique features that make it a useful reagent forin vivo PCA. First, CCF2/AM contains butyryl, acetyl and acetoxymethylesters, allowing diffusion across the plasma membrane where cytoplasmicesterases catalyze the hydrolysis of its ester functionality releasingthe polyanionic (4 anions) β-lactamase substrate CCF2. Because of thenegative charge of CCF2, the substrate becomes trapped in the cell. Inthe intact substrate fluorescence resonance energy transfer (FRET) canoccur between a coumarin donor and fluorescein acceptor pair covalentlylinked to the cephalosporin core. The coumarin donor can be excited at409 nm with emission at 447 nm which is within the excitation envelopeof the fluorescence acceptor (maximum around 485 nm) leading toremission of green fluorescence at 535 nm. When β-lactamase catalyzeshydrolysis of the substrate the fluorescein moiety is eliminated as afree thiol. Excitation of the coumarin donor at 409 nm then emits bluefluorescence at 447 nm whereas the acceptor (fluorescein) is quenched bythe free thiol.

One can perform both positive and negative detection of protein-proteininteractions using the CCF2/AM substrate. For example, positivedetection for a protein-protein interaction consists in observing incells, the conversion of green to blue fluorescence, whereas disruptionof an interaction, a reversion to green fluorescence. This isillustrated by the rapamycin-induced FKBP FRB interaction (FIG. 4). Thecompetitive inhibitor, FK506, disrupts the interaction of theFKBP-rapamycin-FRB interaction. While shown as a FK506concentration-dependent reduction in blue fluorescence, equally it couldbe read as an augmentation of green fluorescence.

The invention is illustrated by the following non-limiting examples.

EXAMPLES Example 1

Materials and Methods

DNA Constructs

GCN4 Zipper Dimerization System

Fragments of β-lactamase (F[1] and F[2]) were amplified by PCR usingProgene (Mendel Scientific) from the ampicillin resistance gene of thevector pQE-32 (Qiagen) with the following oligos: BLF[1]forward:AAAAAAAAGCGG-CCGCACACCCAGAAACGCTGGT; BLF[1] reverse:AAACTCGAGTTA-GCCAGTTAATAGTTTGCG; BLF[2] forward:AAAAAAAAGCGGCCGCACTAC-TTACTCTAGCTTCCC; BLF[2] reverse:AAACTCGAGTTACCAATGCTTAAT-CAGTGAG. The PCR products were introduced atthe 3′ end of a flexible linker of five amino acids(Gly-Gly-Gly-Gly-Ser) in a previously described construct consisting ofGCN4 leucine zipper-5a.a. in pMT3 vector (a eukaryotic expressionvector) resulting in the following constructs: Zip-5a.a.-BLF[1] andZip-5a.a.-BLF[2] with a five amino acids linker.

Example 2

FKBP and FRB Dimerization System

The PCR product were introduced at the 3′ end of a flexible linker offive amino acids (Gly-Gly-Gly-Gly-Ser) in a previously describedconstruct consisting of FRB(FKBP-rapamycin binding domain of FRAP; FRAPis the FKBP-rapamycin binding protein)-5a.a. and FKBP (the FK506 bindingprotein)-5a.a. in pMT3 vector (ref. 10) resulting in the constructsFRB-5a.a.BLF[1] and FKBP-5a.a.-BLF[2] respectively. BLF[1] and BLF[2]correspond respectively to residues 23-197 and 198-286 ofTEM-1β-lactamase.

Example 3 Cell Culture and Transfection

HEK 293 T cells were split 24 h before transfection at 1×10⁵ in 12-wellplates in DMEM (Life Technologies; Grand Island, N.Y.) which wasenriched with 10% cosmic calf serum (HyClone). Cells were transfectedwith the different constructs by using Fugene reagent (Roche) accordingto the manufacturer's instructions.

Example 4

In Vitro Enzymatic Assay with Nitrocefin

48 h after transfection, cells were washed 3 times with cold PhosphateBuffered Saline (PBS) resuspended in 300 μl of cold PBS and kept on ice.Cells were then centrifuged at 4° C. for 30 seconds, the supernatantdiscarded and cells resuspended in 100 μl of cold Phosphate buffer 100mM pH 7.4 (β-lactamase reaction buffer). Cells were lysed with 3 cyclesof freeze and thaw by freezing in dry ice/ethanol for 10 minutes andthawing in a water bath at 37° C. for 10 minutes. Cell membrane anddebris were removed by centrifugation at 4° C. for 5 minutes (10,000×g).The supernatant whole cell lysate was then collected and stored at −20°C. until assays were performed. Assays were performed in 96-well plates(Corning Costar, Cat.no: 3595). For testing β-lactamase activity, 100 μlof Phosphate buffer 100 mM pH 7.4 was aliquoted into each well. To thiswas added 78 μl of H₂O and 2 μl of Nitrocefin 10 mM (final concentrationof 100 μM). Finally, 20 μl of unfrozen cell lysate was added (finalbuffer concentration of 60 mM; final nitrocefin concentration 60 μM).The assays were performed with a Perkin-Elmer HTS 7000 Series Bio AssayReader in absorption mode with the following settings:

A—Measurement mode: Absorption

B—Measurement filter: 492 nm

C—Shaking time: 5 sec

D—Shaking mode: Orbital

E—Shaking intensity: Low

F—Number of flashes: 3

G—Integration start: 0 μs

H—Integration time: 40 μs

I—Number of measurements: 61

J—Length of measurement: 00:20:00

K—Measurement interval: 00:00:20

Example 5

In Vivo Enzymatic Assay and Fluorescent Microscopy with CCF2/AM

HEK 293 cells were cotransfected as described above, and plated onto 6well plates for suspension assays or onto 15 mm glass coverslips (TedPella Inc.) for fluorescence microscopy. 24 h after transfection, cellsare split again to assure 50% confluency the following day (cell densityof 1.5×10⁵). 24 h after splitting the cells were washed 3 times with PBSto remove all traces of serum. Cells were then loaded with thefollowing: 1 μM of CCF2/AM (diluted from a stock 1 mM solution in DMSO)diluted into a physiologic saline solution (HEPES, 10 mM; Sucrose, 6 mM;Glucose, 10 mM; NaCl, 140 mM; KCl, 5 mM; MgCl₂, 2 mM; CaCl₂, 2 mM; pH7.35) for 1 hour. Cells were then washed twice with the physiologicsaline. The cells were resuspended into the same solution and 1×10⁶cells were aliquoted into a 96-well fluorescence white plate (Dynex no7905, VWR Scientific, Cat.no: 62402-980) were read for blue fluorescencewith a Perkin Elmer HTS 7000 Series Bio Assay Reader with the followingsettings:

A. Measurement mode: Fluorescence [RFU] Top

B. Excitation filter: 405 nM

C. Emission filter: 465 nM

D. Gain mode: Manual

E. Gain: 60

A. Number of flashes: 3

B. Lag time: 0 μs

A. Integration time: 40 μs

B. Shaking time: 5 sec

C. Shaking mode: Orbital

D. Shaking intensity: Low

For fluorescence microscopic studies the cells were kept in thephysiologic saline on the 15 mm glass coverslips. Treatment of cellsprior to microscopy was the same as described above unless otherwiseindicated. Fluorescence microscopy was performed on live HEK 293 T cellswith an inverse Nikon Eclipse TE-200 (objective plan fluor 40× dry,numerically open at 0.75) Images were taken with a digital CCD cooled(−50° C.) camera, model Orca-II (Hamamatsu Photonics (expositions for 1second, binning of 2×2 and digitalization 14 bits at 1.25 MHz). Sourceof light is a Xenon lamp Model DG4 (Sutter Instruments). Emissionfilters can be change by a emission filter switcher (model Quantoscope)(Stranford Photonics). Images were visualized with ISee software(Inovision Corporation) on a O2 Silicon Graphics computer.

The following selected filters were used:

The filters used: Filter set #31016 (Chroma Technologies);

Excitation filter: 405 nm (passing band of 20 nm);

Dichroic Mirror: 425 nm DCLP;

Emission filter #1: 460 nm (passing band of 50 nm);

Emission filter #2: 515 nm (passing band of 20 nm).

Example 6

Improving the β-lactamase PCA

Mutational Studies of the Fragments

Several point mutations in full length TEM-1 β-lactamase are known toimprove catalytic activity. These mutations are E104K and M182T forBLF[1] and G238S for BLF[2]. For example, the minimum inhibitoryconcentration for cefotaxime is 20,000 fold higher than the wild-typeTEM-1 and catalytic efficiency (k_(cat)/k_(m)) is 2383 times higher. Twoof these mutations are located in fragment [1] and the other in fragment[2].

Example 7

Positive and Negative Survival Selection Assays

As mentioned in the introduction, TEM-1β-lactamase is a standardantibiotic resistance gene incorporated into most commercial vectors forclonal positive selection. It is obvious then, that a PCA can bedesigned based on the same principal, where positive selection forreassembly of the enzyme by interaction of proteins fused to thefragments would be the basis for survival-selection. The same fragmentsas used in the mammalian assays can be used, while in both cases, the 23amino acid signal peptide sequence would need to be fused at theN-termini of both protein-BLF fusions.

The use of antitumor prodrugs forms the basis for a negative selectionassay in bacteria or mammalian cells¹². The chemistry used in theCCF2/AM strategy has been previously applied to designing cell-specifictargeted antitumor agents. As in CCF2/AM a cytotoxic drug is conjugatedto cephalosporin via a thioether, or other appropriate leaving group.Cells are then treated with a cell-surface antigen-specific antibodyfused to β-lactamases. On encountering β-lactamase, the rearrangementabout the cephalosporin β-lactam ring results in release of thecytotoxic prodrug in an active form. In a realization of a negativeselection assay, the disruption of the interaction between two proteinsthat are fused to the β-lactamase fragments would render cellsexpressing these fusions resistant to treatment with the cytotoxicprodrugs by the prevention of fragment complementation and thusβ-lactamase activity. This approach could be used to screen forcompounds that inhibit a protein-protein interaction.

REFERENCES CITED

1. Christensen, H., Martin, M. T. & Waley, S. G. (1990), Biochem. J.266, 853.

2. Sutcliffe, J. G. (1978), PNAS 75, 3737.

3. Page, M. I. (1987), Adv. Phys. Org. Chem. 23, 165.

4. Matagne, A., Lamotte-Brasseur, J. & Frere, J. M. (1998), Biochem. J.330, 581.

5. Philippon, A., Dusart, J. Joris, B. & Frere, J. M. (1998), CMLS 54,341.

6. Kadonaga, J. T. et al. (1984), J. Biol. Chem. 259, 2149.

7. PDB Structure # : 1TEM or 1AXB

8. O'Callaghan et al. (1972)

9. Zlogarnik, G. et al. (1998), Science 279, 84.

10. Remy, I. & Michnick S. (1999), PNAS 96, 5394.

11. Zaccolo, M. & Gherardi, E. (1999), J. Mol. Biol. 285, 775.

12. Kerr, D. E., Li, Z., Siemers, N. O., Senter, P. D. & Vrudhula, V.M., 1998. Development and activities of a new melphalan prodrug designedfor tumor-selective activation. Bioconjug Chem 9: 255-259; Vrudhula, V.M., Svensson, H. P. & Senter, P. D., 1995. J Med Chem 38: 1380-1385;Senter, P. D., Svensson, H. P., Schreiber, G. J., Rodriguez, J. L. &Vrudhula, V. M., 1995. Bioconjug Chem 6: 389-394; Svensson, H. P.,Wallace, P. M. & Senter, P. D., 1994. Bioconjug Chem 5: 262-267.

Although the present invention has been described with reference tospecific details of certain embodiments thereof, it is not intended thatsuch detail should be regarded as limitations upon the scope of theinvention, except as and to the extent that they are included in theaccompanying claims.

What is claimed is:
 1. An assay method comprising: (A) generating: 1) atleast a first fragment of a reporter molecule linked to a firstinteracting domain and at least a second fragment of a reporter moleculelinked to a second interacting domain, or 2) nucleic acid molecules thatcode for A)1) and subsequently allowing said nucleic acid molecules toproduce their coded products; then, (B) allowing interaction of saiddomains; and (C) detecting reconstituted reporter molecule activity,where said reporter molecule catalyses the hydrolysis of the amide bondof β-lactam rings in penicillin- or cephalosporin compounds.
 2. An assayaccording to claim 1 where said reporter molecule is an enzyme.
 3. Anassay according to claim 1 where said reporter molecule is aβ-lactamase.
 4. An assay according to claim 1 where said reaction withsaid substrate is essentially irreversible.
 5. An assay according toclaim 1, 2, 3, or 4 where said substrate comprises Nitrocefin orCCF2/AM.
 6. An assay according to claim 1, 2, 3, or 4 performed in vivo.7. An assay according to claim 1, 2, 3, or 4 where said reportermolecule is not normally present in eukaryotes.
 8. An assay methodcomprising: (A) exposing a host cell to: 1) at least a first fragment ofa reporter molecule linked to a first interacting domain and at least asecond fragment of a reporter molecule linked to a second interactingdomain; or 2) compounds that code therefor; and (B) detectingreconstituted reporter molecule activity, where a reporter molecule anda host cell are used that yield a signal essentially without anyintrinsic background.
 9. An assay according to claim 1, 2, 3, 4, or 8whose signal to background ratio is about 30:1 or higher.
 10. An assayaccording to claim 1, 2, 3, 4, or 8 where said signal can be observed byeye.
 11. An assay according to claim 10 where said substrate comprisesNitrocefin.
 12. An assay method comprising: (A) allowing at least twomolecules capable of mutual interaction to draw into close molecularproximity at least two reporter molecule fragments which, when in closemolecular proximity, form a complex capable of catalyzing the hydrolysisof the amide bond of β-lactam rings in penicillin- or cephalosporincompounds; and (B) detecting a signal resulting from said reaction. 13.An assay according to claim 12 where said reporter molecule is anenzyme.
 14. An assay according to claim 12 where said reporter moleculeis a β-lactamase.
 15. An assay according to claim 12 where said reactionwith said substrate is essentially irreversible.
 16. An assay accordingto claim 12, 13, 14, or 15 where said substrate comprises Nitrocefin orCCF2/AM.
 17. An assay according to claim 12, 13, 14, or 15 performed invivo.
 18. An assay according to claim 12, 13, 14, or 15 where saidreporter molecule is not normally present in eukaryotes.
 19. An assayaccording to claim 12, 13, 14, or 15 where there is essentially nointrinsic background in the assay.
 20. An assay method comprising: (A)allowing at least two molecules capable of mutual interaction to drawinto close molecular proximity at least two reporter molecule fragmentswhich, when in close molecular proximity, form a complex capable ofcatalyzing the hydrolysis of the amide bond of β-lactam rings inpenicillin- or cephalosporin compounds; and (B) detecting a signalresulting from said reaction, where there is essentially no intrinsicbackground in the assay.
 21. An assay according to claim 12, 13, 14, or15, or 20 whose signal to background ratio is about 30:1 or higher. 22.An assay according to claim 12, 13, 14, or 15, or 20 where said signalcan be observed by eye.
 23. An assay according to claim 22 where saidsubstrate comprises Nitrocefin.
 24. An assay according to claim 12, 13,14, or 15, or 20 where said reaction occurs with a cell and saidsubstrate becomes trapped within said cell after entrance therein. 25.An assay method comprising: (A) allowing at least two molecules capableof mutual interaction to draw into close molecular proximity at leasttwo reporter molecule fragments which, when in close molecularproximity, form a complex capable of catalyzing the hydrolysis of theamide bond of β-lactam ring in penicillin- or cephalosporin compounds;and (B) detecting a signal resulting from said reaction, where saidreaction occurs with a cell and said substrate becomes trapped withinsaid cell after entrance therein.
 26. An assay according to claim 12,13, 14, or 15, or 20 where a reporter molecule substrate is added thathas a fluorescent signal-producing system covalently associatedtherewith.
 27. An assay method comprising: (A) allowing at least twomolecules capable of mutual interaction to draw into close molecularproximity at least two reporter molecule fragments which, when in closemolecular proximity, form a complex capable of catalyzing the hydrolysisof the amide bond of β-lactam rings in penicillin- or cephalosporincompounds; and (B) detecting a signal resulting from said reaction,where a reporter molecule substrate is added that has a fluorescentsignal-producing system covalently associated therewith.
 28. An assayaccording to claim 27 where said reaction results in a change influorescent signal production.
 29. An assay according to claim 27 wherea compound is added that leads to a detectable decrease in reportermolecule activity by interfering with said mutual interaction.
 30. Acellular assay method comprising: (A) allowing at least two moleculescapable of mutual interaction to draw into close molecular proximity atleast two reporter molecule fragments which, when in close molecularproximity, form a complex capable of catalyzing the hydrolysis of theamide bond of β-lactam rings in penicillin- or cephalosporin compounds;and (B) detecting cell survival as an indication of said reaction. 31.An assay according to claim 30 where a compound capable of interferingwith said mutual interaction is added.